Evidence for the two-step binding of ATP to myosin subfragment 1 by the rapid-flow-quench method

Abstract

1. The initial steps on the myosin ATPase (EC 3.6.1.3) pathway are taken to be: (formula; see text) A two-step binding for ATP is assumed, but the evidence for it is unconvincing; because of the rapidity of the process unambiguous values for K1 and K2 are not available. 2. We investigated the myosin mechanism by the chemical flow-quench technique. Reaction mixtures containing [gamma-32P]ATP plus myosin subfragment 1 were quenched in unlabelled ATP (ATP chase) or acid (Pi burst). 3. We show that the ATP-chase method can lead directly to unambiguous values for K1 and k+2. 4. The binding process was slowed down by 40% ethylene glycol. It was studied as a function of the ATP concentration. A limiting plateau resulted, showing a two-step binding for ATP, and values for K1 and k+2 were obtained. 5. K1 and k+2 are rather sensitive to the experimental conditions. Ethylene glycol and lowering of the pH decrease both constants, but an increase in KCl concentration increases them. This suggests that the binding of ATP to myosin is of an electrostatic nature. 6. The Pi-burst method can lead directly to k+3 + k-3, but under certain conditions the kinetics are governed by K1 and k+2. This uncertainty of the interpretation of Pi-burst experiments is discussed.

The limiting rate of the ATP‐mediated dissociation of actin from rabbit skeletal muscle myosin subfragment 1

“…In this respect it is of interest to consider the effect of ethylene glycol on the rate of ATP-binding to both Sl and actoS1, The results presented here show that ethylene glycol has only a small effect on the observed rate of ATP-induced dissociation of actoS 1. This is in marked contrast to the results in [14] where it was found that in low salt (5 mM KCI), ATP-binding to Sl alone is markedly affected by ethylene glycol. Basing their analysis on the two-step binding model in [22], the authors found that Kr (the rapid equilibrium of the initial Sl-ATP complex) increases from 5 x 10' M-' to 1.25 x lo5 M-' in the presence of 40% ethylene glycol, and k+z (the rate of isomerisation of the binary complex) decreases from 160 s-' to 16 s-'.…”

“…actoS1 remains very fast in ethylene giycoi which contrasts with the situation for Sl alone where the rate of ATP-binding is markedly reduced. At 15'C in low salt the rate of ATPbinding to Sl is reduced from about 160 s-l in aqueous solution 1131 to 16 s-r in 40% ethylene glycol [14], whereas the rate of ATPinduced dissociation of actoS1 remains > 1000 s-r in both solvents. A change in slope of an Arrhenius plot can be caused either by a change in the rate-limiting step of a process, or by a phase change in the system affecting the protein, the substrate or the solvent.…”

A transient kinetic study of enthalpy changes during the reaction of myosin subfragment 1 with ATP.

“…This cannot be measured as accurately as the nucleotide concentration, since there is always an inactive fraction of SI. Chemical measurements of ATP binding give values of 70-85 % activity (Barman et al, 1983); a value of 80% was assumed for our experiments. The results of such an experiment are shown in Fig.…”

Dimethyl sulphoxide enhances the effects of Pi in myofibrils and inhibits the activity of rabbit skeletal muscle contractile proteins

“…Lowering the temperature has also provided valuable information, since the kinetics of some of the steps are affected more than others [14,15]. Ethylene glycol, which is used as an anti-freeze in most studies carried out at subzero temperatures, also modifies catalysis and cross-bridge kinetics in its own right, without changing structure or mechanism in any fundamental way [15][16][17][18][19][20][21][22]. It has been particularly valuable in providing access to the very fast reactions involving ATP binding, isomerization and actin dissociation at the beginning of the cycle [20,[23][24][25].…”